Children with Acute Lymphoblastic Leukemia (ALL) diagnosed with resistant
phenotypes and those who relapse have a dismal prognosis for cure. In
search for novel treatment strategies, we identified the AMP activated
protein kinase (AMPK) as a potential drug target based on its effects on
cell growth and survival. We have shown previously that AICAR-induced
AMPK activation also induced a compensatory survival mechanism via
PI3K/Akt signaling.

Results

In the present study, we further investigated the downstream signaling
induced by AMPK activation in ALL cells. We found that AICAR-induced
AMPK activation resulted in up-regulation of P-Akt (Ser473 and Thr308)
and decrease of P-mTOR (Ser2448) expression and downstream signaling. We
determined that activation of P-Akt (Thr308) was mediated by
AMPK-induced IGF-1R activation via phosphorylation of the insulin
receptor substrate-1 (IRS-1) at Ser794. Inhibition of IGF-1R signaling
using the tyrosine kinase inhibitor HNMPA(AM)3 resulted in
significant decrease in P-IRS-1 (Ser794) and P-Akt (Thr308).
Co-treatment of AICAR plus HNMPA(AM)3 prevented AMPK-induced
up-regulation of P-Akt (Thr308) but did not alter the activation of
P-Akt (Ser473). Inhibition of AMPK using compound-C resulted in
decreased P-Akt expression at both residues, suggesting a central role
for AMPK in Akt activation. In addition, inhibition of IGF-1R signaling
in ALL cells resulted in cell growth arrest and apoptosis. Additional
Western blots revealed that P-IGF-1R (Tyr1131) and P-IRS-1 (Ser794)
levels were higher in NALM6 (Bp-ALL) than CEM (T-ALL), and found
differences in IGF-1R signaling within Bp-ALL cell line models NALM6,
REH (TEL-AML1, [t(12;21)]), and SupB15 (BCR-ABL, [t(9;22)]). In these
models, higher sensitivity to IGF-1R inhibitors correlated with
increased levels of IGF-1R expression. Combined therapy simultaneously
targeting IGF-1R, AMPK, Akt, and mTOR pathways resulted in synergistic
growth inhibition and cell death.

Acute Lymphoblastic Leukemia (ALL) is the most common hematological malignancy
affecting children and adolescents, and remains the leading cause of
cancer-related mortality in this age group [1]. ALL is a heterogeneous disease with distinct phenotypes
segregated by the presence of non-random translocations and genomic deletions
and amplifications [2]. Despite
significant progress in the treatment of ALL, a large number of children
continue to relapse and for them, outcome remains poor. In addition, adults are
generally diagnosed with resistant phenotypes of ALL and continue to respond
poorly to existing treatment regimens. Therefore, novel therapies need to be
developed. Recently, our laboratory identified AMP activated protein kinase
(AMPK) as a potential target for ALL therapy due to its effects on cell growth
and its signaling crosstalk with critical metabolic and oncogenic pathways
[3]. Treatment with the AMPK activator
5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) induced
apoptotic cell death in ALL cells mediated by AMPK, mTOR, P27, P53, and p38-MAPK
[3]. In addition, AICAR significantly
increased P-Akt (Ser473) following AMPK activation and mTOR down-regulation,
which was viewed as a compensatory survival mechanism. Akt (protein kinase B) is
involved in critical survival pathways, and inhibits apoptosis via
phosphorylation of the pro-apoptotic protein BAD at Ser136, which prevents its
inhibitory association with the anti-apoptotic Bcl-2 protein [4-6].
Akt is activated by phosphorylation of two key residues: Thr308 within the
T-loop of its catalytic domain, and Ser473 located in the hydrophobic region of
its C-terminal domain [7,8]. Phosphorylation of both residues is
essential for maximal activity [8] and was
found to be regulated by independent mechanisms [9]. Phosphorylation of Akt at Ser473 involves rictor, a member of
the TORC2 complex known to modulate the activity of mTOR [7,10-12], while phosphorylation of Thr308 is
mediated by PDK1 and PIP3 following phosphorylation of PIP2 by PI3K [13,14]. The latter mechanism is responsible for the described feedback
loop inhibition of Akt phosphorylation mediated by mTOR-dependent
phosphorylation of IRS-1 at Ser312, the immediate downstream effector protein of
the insulin-like growth factor-1 receptor (IGF-1R) [15,16].
Phosphorylation of IRS-1 (Ser312) by P-mTOR promotes conformational changes and
subsequent detachment from the receptor and degradation [17], and inhibits potentiation of Akt by IGF-1R/IRS-1
signaling [18]. Conversely, inhibition of
mTOR results in IRS-1 activation and increased phosphorylation of Akt at Thr308
[19].

IGF-1R is one of four transmembrane receptors (IGF-1R, IGF-IIR, IR, and hybrids
receptors of IGF and IR) that compose the IGF-1R signaling system in addition to
the three circulating ligands (IGF-I, IGF-II, and insulin) and multiple
regulatory IGF-binding proteins (IGFBP-1 to -6) [20-23]. IGF-1R is
ubiquitously expressed in human cancer cells compared to normal tissues [24]. Elevated plasma concentrations of
IGF-1, IGFBP-2, and IGFBP-3 have been linked to more aggressive phenotypes in
breast, colon, prostate, lung cancer, and ALL [25,26]. IGF-1R exerts its
action through activation of downstream signaling cascades that regulate
metabolic and oncogenic pathways important for cellular growth [27]. IGF-1R signaling has been linked to
the regulation of normal and malignant hematopoietic cells. Significant
differences in the expression of the IGF-1 system components IGF-II, IGFBP-2,
IGFBP-4 and IGFBP-5 have been found between B-lineage and T-lineage ALL [28-30]. Taken together, this suggests that activation of IGF-1R
signaling and its downstream pathways may confer ALL cells a survival advantage
by influencing growth and metabolic adaptations aimed at supporting accelerated
growth. Therefore, to delineate the mechanism responsible for ALL cell survival
regulated by AMPK and IGF-1R and to understand the role of IGF-1R in this
process, we investigated the relationship between AMPK and the cell
proliferation and survival pathways downstream of IGF-1R/IRS-1. As a result, we
uncovered potential combination therapies that simultaneously target key factors
within these signaling cascades.

Recently, we reported that treatment of ALL cell lines with AICAR induced
growth inhibition and apoptosis, and resulted in increased expression of
P-Akt (Ser473) [3]. Phosphorylation of
Akt, especially at the Ser473 residue, has been shown to be regulated by the
mTOR/TORC2 complex [7,10-12], whereas phosphorylation of Akt at Thr308 was shown to be
regulated by mTOR but through a feedback loop inhibition mechanism targeting
IRS-1 [15-17]. To investigate the role of AMPK and mTOR in this
process, we examined the levels of P-mTOR (Ser2448) and P-IRS-1 (Ser794, a
residue known to be phosphorylated by AMPK) [31] in CCRF-CEM (T-ALL) and NALM6 (Bp-ALL) cells treated with
AICAR. As expected, levels of P-AMPK and P-Akt (Ser473) were increased
following treatment with AICAR (200 and 500 μM), while expression of
P-mTOR (Ser2448) was significantly decreased (p < 0.001,
for control vs. AICAR treated cells) (Fig. 1). Concomitantly, expression of P-IRS-1
(Ser794) was significantly increased in a dose dependent manner (p
< 0.05, for control vs. AICAR treated cells).
These changes in phosphorylated protein expression directly correlated with
level of P-AMPK (Thr172), and inversely correlated with the degree of P-mTOR
down-regulation (Fig. 1). These data
indicate that the compensatory increase in P-Akt expression seen in
AICAR-treated ALL cells results from both activation of IRS-1 by AMPK, and
inhibition of the mTOR mediated feedback loop inhibition of IRS-1 activity.
Nevertheless, as previously demonstrated, this compensatory up-regulation of
P-Akt was unable to rescue ALL cells from apoptotic death following
AICAR-induced AMPK activation [3].

Figure 1

Activation of AMPK induces phosphorylation of IRS-1 at Ser794
and down-regulation of mTOR (Ser2448) in ALL cell lines.
Western blot analysis of the expression of AMPK (Thr172), Akt
(Ser473), mTOR (Ser2448) and IRS-1 (Ser794) in CCRF-CEM (T-ALL) and
NALM6 (Bp-ALL) cells treated with AICAR (200 and 500 μM) and
incubated for 24 h at 37°C. The density value of each band was
normalized to β-actin level and expressed relative to control
(shown as fold induction).

AICAR-induced phosphorylation of Akt at Ser473 is independent of
IGF-1R/IRS-1 signaling in ALL but requires AMPK activation

To characterize the extent to which the increase in P-Akt expression was
dependent on IGF-1R/IRS-1, we used the specific tyrosine kinase inhibitor
HNMPA(AM)3 (IGF1Ri) to inhibit IGF-1R/IRS-1 signaling, and
examined its effects on P-IRS-1 (Ser794) and P-Akt (Ser473 and Thr308)
expression in AICAR-treated CCRF-CEM and NALM6 cells using Western
immunoblotting. As shown in Fig. 2,
treatment with AICAR (200 μM) alone for 24 h increased the expression
of P-IRS-1 (Ser794) and P-Akt at Ser473 and Thr308 by over two fold, whereas
treatment with HNMPA(AM)3 alone (IGF1Ri, 10 μM) decreased
significantly the phosphorylation of P-IRS-1 (Ser794) and P-Akt (Thr308)
(p < 0.0001, for P-IRS-1 expression in control
vs. HNMPA(AM)3 treated cells; p
< 0.001, for P-Akt expression in control vs.
HNMPA(AM)3 treated cells), but had a negligible effect on
P-Akt (Ser473). More important, co-treatment with an IGF-1R inhibitor in
cells exposed to AICAR failed to restore the observed AICAR-induced
up-regulation of P-IRS-1 (Ser794), and P-Akt (Thr308), while phosphorylation
of Akt at Ser473 remained unaffected (Fig. 2,
A + IGF1Ri). These findings indicate that AICAR-induced Akt
phosphorylation at Thr308 is dependent of IGF-1R/IRS-1 activation whereas
phosphorylation of Akt at Ser473 occurs independently of IGF-1R/IRS-1
signaling but requires AMPK activation. Therefore, AMPK activation by AICAR
promotes activation of Akt by two mechanisms: phosphorylation of Akt
(Thr308) by IGF-1R/IRS-1 (Ser794) signaling mediated by AMPK and its
downstream down-regulation of mTOR, and the other through phosphorylation of
Akt (Ser473) by an AMPK-dependent mechanism.

Figure 2

AICAR-induced AMPK activation phosphorylates Akt at Ser473 in a
mechanism independent of IGF-1R/IRS-1 signaling in ALL cell
lines. Western blot analysis of P-IRS-1 (Ser794), P-Akt
(Ser473), and P-Akt (Thr308) in CCRF-CEM and NALM6 cells treated
with 0.1% DMSO (CTRL), AICAR (200 μM), the IGF-1R tyrosine
kinase inhibitor HNMPA(AM)3 (IGF1Ri, 10 μM), or
both agents (A + IGF1Ri) and incubated for 24 h at 37°C. The
levels of P-IRS-1 and P-Akt were normalized to β-actin
(loading control) and expressed relative to control (shown as fold
induction).

To further investigate the role of AMPK in the activation of Akt, we compared
the effects of the AMPK activator AICAR and compound-C, a known specific
inhibitor of AMPK [32,33]. Western blot analysis of protein
extracts from CCRF-CEM and NALM6 cells treated with either AICAR (100 &
200 μM) or compound-C (2.5 & 5.0 μM) showed that activation
of AMPK correlated with phosphorylation of Akt at both residues (Ser473 and
Thr308), and conversely inhibition of AMPK by compound-C also led to
down-regulation P-Akt at both residues (Fig. 3). To ascertain the affects of P-AMPK in these experiments, the
functional activation or inhibition of AMPK signaling were confirmed by the
determining the phosphorylation status of P-ACC (Ser79). As seen in Fig.
3, expression of P-ACC directly
correlated with the phosphorylation status of AMPK at Thr172. These data
together with data presented in Fig. 2,
strongly suggest that functional AMPK signaling is required for activation
of Akt at both Ser473 and Thr308, but the phosphorylation of Akt at Thr308
also requires IGF-1R/IRS-1 signaling. Therefore, the compensatory activation
of Akt seen in ALL cells following AICAR-induced AMPK activation resulted
from phosphorylation of Akt at Thr308 and Ser473 (Fig. 2).

Figure 3

Functional AMPK activity is required for activation of Akt at
Ser473 and Thr308 in ALL cell lines. CCRF-CEM and NALM6
cells were exposed to either the AMPK activator AICAR (100 and 200
μM) or the AMPK inhibitor compound-C (CompC, 2.5 and 5.0
μM) for 24 h at 37°C. Proteins were extracted and
analyzed by Western immunoblotting for the expression of P-Akt
(Ser473 and Thr308), P-AMPK (Thr172), and P-ACC (Ser79). The density
value of each band was normalized to β-actin level and
expressed relative to control (shown as fold induction).

Phosphorylation of Akt at Thr308 was shown to be sufficient to induce Akt's
pro-survival effects [34] but
phosphorylation of both residues is needed for optimal activity. To examine
the role of IGF-1R/IRS-1 signaling in ALL cell survival, we evaluated the
effects of IGF-1R inhibition using HNMPA(AM)3 (2 - 100 μM)
on cell growth and apoptosis using a panel of ALL cell models. As shown in
Fig. 4A, treatment of CCRF-CEM and
NALM6 cells with HNMPA(AM)3 inhibited their growth in a
dose-dependent manner with calculated EC50 values of 16.5 μM and 6.1
μM for CCRF-CEM and NALM6, respectively. We then extended our analysis
to other Bp-ALL subtypes characterized by the non-random translocations REH
[t(12;21)] and SupB15 [t(9;22)]. In NALM6 treatment with HNMPA(AM)3
(10 μM) led to 50% growth inhibition compared to 40% and 25% in
REH and SupB15 cells, respectively (Fig. 4B).

Figure 4

Inhibition of IGF-1R tyrosine kinase activity induces growth
inhibition and apoptosis in ALL cell lines. Cell growth
(A) and level of apoptosis (C)
detected in ALL CCRF-CEM and NALM6 cells treated with the IGF-1R
inhibitor HNMPA(AM)3 (2 - 100 μM) and incubated for
24 h at 37°C. Proliferation (B) and level of
apoptosis (D) detected in Bp-ALL subtypes NALM6, REH
(t[12;21]), and SupB15 (t[9;22]) treated with HNMPA(AM)3
(10 μM) and incubated for 24 h at 37°C. The cell
growth (viability) values are expressed as a percentage relative to
those obtained with untreated control cells (mean ± SEM, n =
3). Annexin V-FITC/PI staining data (apoptosis) were normalized and
expressed as fold induction relative to control values (mean ±
SEM, n = 3) as described in Methods.

To determine if IGF-1R inhibition was cytostatic or cytotoxic in ALL cells,
we determined induction of apoptosis in these same cell models. CCRF-CEM and
NALM6 cells were treated with increasing concentrations of HNMPA(AM)3
(2 - 100 μM) and apoptosis was assayed using Annexin V-FITC/PI
staining. Fig. 4C shows that
HNMPA(AM)3 induced apoptotic cell death in a dose-dependent
manner in NALM6, and to a lower extent in CCRF-CEM cells. Comparatively, the
maximal fold increase in apoptotic cell death was approximately 40-fold
compared to control in NALM6 cells, whereas only a 10-fold increase in
apoptotic death was observed in CCRF-CEM cells (Fig. 4C). Level of apoptosis in the Bp-ALL subtypes REH
[t(12;21)] and SupB15 [t(9;22)] following treatment with HNMPA(AM)3
(10 μM) was significantly lower compared to NALM6 cells
(p = 0.0032, for NALM6 vs. REH;
p = 0.0016, for NALM6 vs. SupB15). REH
and SupB15 cells exhibited only a 2-fold increase in apoptotic cell death
compared to a 6-fold increase in NALM6 cells (Fig. 4D). Similar fold differences were observed over a range
of drug concentrations. Interestingly, the translocation t(9;22) encoding
for the BCR-ABL fusion protein expressed in SupB15 cells was shown to induce
autocrine IGF-1 signaling in leukemia, which may confer clinical resistance
due to higher IGF-1R signaling and constitutive P-Akt activity. Taken
together, these data raise the intriguing possibility that cell lineage of
origin (B- vs. T-ALL) and the presence of non-random
translocations may modulate IGF-1R activity and consequently may influence
ALL cell death vs. cell survival when exposed to IGF-1R
inhibitors.

Differential expression level of IGF-1R and downstream signaling factors
in ALL cells

The different levels of sensitivity to the IGF-1R inhibitor observed among
CCRF-CEM (T-ALL) and NALM6 (Bp-ALL) cells, and within Bp-ALL REH and SupB15
subtypes expressing selected non-random translocations prompted us to
investigate the mechanism underlying these differences. To address this
question, we performed Western blot analysis of key factors associated with
the IGF-1R signaling cascade in these cell models. As shown in Fig. 5A, NALM6 cells expressed higher levels
of phospho-IGF-1R (Tyr1131) and phospho-IRS-1 (Ser794) than CCRF-CEM cells.
Similarly, lower levels of expression of P-IGF-1R (Tyr1131) and P-IRS-1
(Ser794) were detected in the Bp-ALL REH [t(12;21)] and SupB15 [t(9;22)]
subtypes characterized by non-random translocations in comparison to NALM6
(Fig. 5B). In addition, the expression
of P-Akt was higher in CCRF-CEM cells (shown in Fig. 3) and REH cells (Fig. 5B), which correlated with these cell models having either a
mutation or a deletion in the PTEN gene, respectively [35,36].
Similarly, the high level of P-Akt found in SupB15 cells (carrying the
BCR-ABL gene fusion) results from inhibition of PP1α, a serine
phosphatase that negatively regulates the PI3K/Akt pathway [37]. Mechanistically, higher levels of
IGF-1R/IRS-1 expression correlated with higher sensitivity to IGF-1R
inhibition, with NALM6 cells exhibiting the highest expression of IGF-1R and
the highest sensitivity to apoptotic cell death following IGF-1R inhibition.
As expected, treatment with the IGF-1R inhibitor HNMPA(AM)3
(IGF1Ri, 10 μM) reduced considerably the expression of both
IGF-1R and IRS-1 phosphorylated proteins (Fig. 5A). Additionally, phosphorylation of IRS-1 at Ser312, the
residue targeted by mTOR and responsible for the negative feedback-loop
inhibition, was inversely expressed compared to the expression of P-IGF-1R
(Tyr1131) in all the cells examined (Fig. 5A and 5B). The activity of
P-mTOR was monitored using P-4EBP1 (Thr70) expression, its immediate
downstream target [38], and
demonstrated that mTOR activity was down-regulated in all cell lines
following IGF-1R inhibition. These data further suggest that "addiction" of
the cells to IGF-1R activity as determined by P-IGF-1R (Tyr1131) and P-IRS-1
(Ser312) expression makes cells more dependent on IGF-1R signaling for
survival, and therefore more susceptible to IGF-1R inhibition.

Our laboratory and others have demonstrated that significant functional
cross-talk between AMPK, mTOR, IGF-1R/IRS-1, and Akt signaling factors occur
in leukemia cells [3,9,39-42]. Since inhibition
of IGF-1R activity is capable of inducing growth inhibition and apoptotic
cell death, we reasoned that co-targeting these interconnected pathways
would result in enhanced cytotoxicity. To test this hypothesis we tested
three combination strategies in ALL cell line models. First, we evaluated
agents targeting simultaneously the AMPK (AICAR, 100 μM) and IGF-1R
(HNMPA(AM)3, 1 μM) signaling proteins. This combination
resulted in significant growth inhibition (p < 0.001,
for AICAR + HNMPA(AM)3 vs. control, AICAR alone,
and HNMPA(AM)3 alone) in CCRF-CEM and NALM6 cell lines examined
(Fig. 6A) with a calculated combination
index (CI) of 0.47 and 0.55 for CCRF-CEM and NALM6, respectively. Second, we
tested whether inhibition of Akt, downstream to IGF-1R signaling, in the
presence of AICAR would also increase growth inhibition. As shown in Fig.
6B, combination of AICAR (200
μM) plus the Akt inhibitor X (AIX, 9 μM) had similar effects
with CI values of 0.90 and 0.85 for CCRF-CEM (p < 0.001,
for AICAR + AIX vs. control, AICAR alone, and AIX alone)
and NALM6 (p < 0.05, for AICAR + AIX
vs. control, AICAR alone, and AIX alone), respectively.
These results suggest that blocking activation of Akt by either inhibiting
IGF-1R/IRS-1 activity or the downstream interference with Akt
phosphorylation, greatly increases the growth inhibition when AMPK is
simultaneously activated by AICAR in ALL cells. Third, we tested the
combined inhibition of IGF-1R and mTOR, which is negatively regulated
following AMPK activation [3]. For
these experiments we treated cells with the mTOR inhibitor rapamycin plus
the IGF-1R inhibitor HNMPA(AM)3. Although blocking mTOR activity
would have a negative effect on cell proliferation secondary to inhibition
of protein synthesis, it would also relieve the feedback loop inhibition on
IRS-1 activating Akt, which might promote cell growth. As presented in Fig.
6C, treatment of CCRF-CEM and NALM6
cells with rapamycin (1 μg/ml) and HNMPA(AM)3 (0.5 - 1.0
μM) induced growth inhibition with CI values of 0.41 and 0.88 for
CCRF-CEM and NALM6 cells, respectively. Therefore, the three combination
strategies tested resulted in synergistic growth inhibition in both cell
lines examined, as evidenced by CI values <1 in all cases.

We then analyzed induction of the cell death resulting from these drug
combinations and found that only the combination AICAR plus AIX, targeting
AMPK and Akt simultaneously, was synergistic with a CI value of 0.89 and
0.78 for CCRF-CEM and NALM6 cell lines, respectively (Fig. 7B). Although additional cell death was
observed for the other combinations as compared to single drug alone, none
of the cytotoxic effects resulting from the two other drug combinations were
synergistic (Fig. 7A and 7B). The combination HNMPA(AM)3
plus AICAR resulted in a "borderline" CI of 0.99 and was considered
additive, whereas the combination of HNMPA(AM)3 plus rapamycin was found to
be antagonistic with CI >1. In both cases in which combination therapy
was either additive or synergistic in inducing cell death in NALM6 and
CCRF-CEM cells, activation of AMPK signaling was co-targeted. These data
suggest that the master energy regulator AMPK plays a pivotal role in
triggering apoptotic cell death when these signaling cascades are
co-targeted, and that the cross-talk between AMPK and the IGF-1R, Akt and
mTOR pathways appears to be important in determining cellular fate following
perturbations of these cascades. Taken together, our data indicate that
blocking simultaneously both the key cell proliferation regulator mTOR, and
the IGF-1R-induced Akt phosphorylation pathway resulted in significant cell
growth inhibition and cell death by interfering with the mechanism of cell
survival triggered by treatment with single agents.

Figure 7

Co-targeting AMPK and Akt signaling pathway induces synergistic
cell death in ALL cell lines. Cell death values obtained
from the ALL CCRF-CEM and NALM6 cells shown in Fig. 6 which were
treated with either AICAR (100 μM) plus the IGF-1R inhibitor
HNMPA(AM)3 (1.0 μM) (panel A);
AICAR (200 μM) plus the Akt inhibitor-X (AIX, 9.0 μM)
(panel B); or rapamycin (1.0 μg/ml) plus
HNMPA(AM)3 (0.5 and 1.0 μM) (panel
C). The cell death values were generated from the
trypan blue exclusion data and are expressed as a percentage
relative to those obtained with control cells (mean ± SEM, n =
3). Combination index (CI) values were determined for each drug
combination as described in Methods (C <1, = 1, and >1
indicate synergism, additive effect, and antagonism,
respectively).

Discussion

In search for novel treatment strategies, we investigated AMPK signaling as
potential target for ALL therapy. Our results, together with our previously
published report [3] reveal that
activation of AMPK by AICAR induces a compensatory survival response through
activation of Akt at both of its functional residues Ser473 and Thr308. Although
phosphorylation of Akt at both residues is critical for maximum catalytic
activity [9,43], it has been established that phosphorylation of Thr308
is sufficient to activate its kinase activity and support cell survival [34]. We show that the mechanism of Akt
activation in ALL cells is mediated in part by AMPK-induced phosphorylation of
IRS-1 at Ser794, the immediate downstream effectors of the IGF-1R signaling
cascade, and also in part by AMPK-induced inhibition of mTOR and its downstream
feedback loop inhibition of IRS-1 (Ser312). Direct interaction between P-AMPK
(Thr172) and phosphorylation of IRS-1 at Ser794 has been shown to occur in
several systems such as cell lines [31,44,45], and insulin-resistant animal models [46], but the biological relevance of this
phosphorylation event is still not clear. Different functions have been reported
for AMPK-induced IGF-1R phosphorylation with some reporting a positive effect on
PI3K/Akt signaling [31] whereas others
reported a negative effect [45-47]. Additive activation of AMPK and Akt
has been shown to regulate important biological functions such as angiogenesis
and glucose metabolism [48,49], suggesting that positive interactions
exist between AMPK and Akt as we report here. Other reports demonstrated that
Akt could negatively regulate AMPK activity by direct binding and
phosphorylation of AMPK at Ser485 [50-52]. These opposite
effects reflect the complexity of the signaling cross-talk that exists between
AMPK, IRS-1, and downstream activation of Akt.

It is clear from our studies that phosphorylation of Akt at Thr308 in
AICAR-treated ALL cells occurs via direct AMPK down-regulation of mTOR and
activation of the IGF-1R/IRS-1 signaling cascade. This compensatory mechanism
promotes cell survival because inhibition of IGF-1R activity in either presence
or absence of AICAR decreases P-IRS-1 (Ser794) and P-Akt (Thr308) levels and
significantly increases apoptotic cell death. The signaling cascade triggered by
activation of tyrosine kinase receptor leading to phosphorylation of IRS-1 and
subsequent activation Akt at Thr308 have been extensively studied and is
mediated by the downstream PI3K and PDK1 kinases [16,34,53]. Additionally, we demonstrate that
phosphorylation of Akt is also dependent on AMPK since inhibition of AMPK
activity with compound-C clearly decreased P-Akt at both residues. AMPK has been
shown to inhibit mTORC1 activity by two different mechanisms: one through
activation of the TSC2, which promotes downstream inhibition of the mTOR
activator Rheb [9,54], and the other through direct phosphorylation of Raptor
at Ser792 blocking mTORC1 activation [55]. Additional studies demonstrated that phosphorylation of Akt at
Ser473 was mediated by mTORC2, a complex formed by the association of rictor,
mSin1, mLST8, with mTOR [7,10,12,34,56,57]. Among mTORC1
and mTORC2, mTOR is the only critical factor that is shared by both complexes
[9]. Thus, it is tempting to speculate
that by down-regulating mTORC1, AMPK could increase the availability of mTOR and
favor the formation of mTORC2, which would promote phosphorylation of Akt at
Ser473. A similar mechanism was proposed for activation of Akt by AMPK in
macrophages expressing a constitutively active form of AMPK [58]. Nevertheless, we can not rule out the
possibility that a distinct mechanism independent of mTORC2 might be involved in
this process.

The data presented herein shows that activity of IGF-1R/IRS-1 was higher in NALM6
vs. CCRF-CEM cells, and that their expression also differs
within Bp-ALL REH and SupB15 subtypes characterized by the non-random
translocations t[12;21], and t[9;22]. More important, these differences
correlated with reduction in P-IRS-1 (Ser794) and P-Akt (Ser473 and Thr308), and
degree of induction of apoptotic death resulting from the pharmacological
inhibition of IGF-1R. Our results raise the intriguing possibility that cell
lineage of origin and/or presence of selected non-random translocations may
influence response to therapy in ALL cells treated with inhibitors of IGF-1R.
This possibility needs to be investigated using primary samples from patients
with ALL. It is also possible that the level of Akt activation in these cells
may also dictate their degree of sensitivity to IGF-1R inhibition. For instance,
it is well known that the CCRF-CEM cell line carries a mutation inactivating
PTEN [35] and that REH cells born a PTEN
deletion [36], both leading to increased
reliance on Akt signaling for cell survival. In addition, SupB15 cells express
high levels of P-Akt because the expression of the BCR-ABL gene fusion inhibits
PP1α, a serine phosphatase that negatively regulates the PI3K/Akt pathway
[37]. Interestingly, the expression
level of P-Akt was the lowest in NALM6 cells which was also the most sensitive
to the IGF-1R inhibitor HNMPA(AM)3 as compared to all of the other
cell lines examined, therefore suggesting that Akt provides a mechanism to
escape cell death following IGF-1R inhibition.

To further assess whether IGF-1R signaling may be influenced by biological
pathways closely linked to cell lineage and non-random chromosomal
translocations, we have mined existing gene expression databases from childhood
ALL patients http://www.stjuderesearch.org/data/ALL1, and found that the
expression of relevant IGF-1 regulatory carriers such as IGFBP2 and IGFBP4
appear to be significantly differentially expressed in ALL in a phenotype
specific manner. The known correlation between these carriers and IGF-1
suggested to us that differences in IGF-1 signaling may exist in ALL, and impact
critical oncogenic and survival signaling pathways. Interestingly, IGF-1R
signaling has been linked to cell lineage of origin in ALL. For instance,
significant differences in the expression of the IGF-1 system components IGF-II,
IGFBP-2, IGFBP-4 and IGFBP-5 have been described between B-lineage and T-lineage
ALL [28-30]. IGFBP-2 was identified as the major regulatory carrier in
childhood leukemia and exhibited an inverse correlation with IGF-1 levels [59], suggesting that activation of IGF-1R
signaling may confer ALL cells a survival advantage and influence induction of
apoptosis. Emerging literature suggests that IGF-1R signaling may also be
influenced by non-random translocations in ALL [60,61]. For instance,
leukemia cells expressing the translocation t(9;22) encoding for the BCR-ABL
fusion not only exhibit a higher degree of resistance to chemotherapeutic drugs
but also were shown to induce autocrine IGF-1 signaling. Thus, it is clear that
IGF-1R pathway may provide ALL cells a survival advantage through its crosstalk
with other critical metabolic networks.

The identification of potential cross-talk within compensatory survival pathways
in ALL cells prompted us to developed simultaneous co-targeting strategies to
induce cell death in ALL cells. We demonstrated that co-targeting IGF-1R and
downstream pathways (AMPK & IGF-1R, mTOR & IGF-1R, and AMPK & Akt)
led to synergistic growth inhibition in ALL cell models. This is consistent with
the study of Bertrand et al. [62] that demonstrated that blocking IGF-1R activity using an
antibody synergized with inhibitors of PI3K/Akt/mTOR pathway by suppressing the
IGF-1R-induced Akt phosphorylation, and consequently promoted apoptosis in
hematopoietic cells. Among the three drug combinations tested, only the one
co-targeting AMPK and Akt resulted in synergistic induction of cell death. This
can be explained in part by differences in the mechanism of action between AIX
vs. HNMPA(AM)3, with AIX being more effective in
inactivating Akt. Taken together, rationally designed simultaneous targeting of
key factors within the AMPK, IGF-1R, and mTOR pathways leads to synergistic
induction of cell growth inhibition by blocking compensatory survival responses
triggered by treatment with single agents. Nevertheless, of the combinations
strategies tested only co-targeting AMPK plus Akt lead to synergistic induction
of apoptosis.

Conclusions

We conclude that IGF-1R and its downstream metabolic and oncogenic pathways
contribute to cell survival and are important to determine pro- or
anti-apoptotic responses in ALL cells to treatment with inhibitors of these
signaling pathways. Our data suggest that PTEN status, AMPK and Akt signaling,
and possibly cell-lineage and non-random translocations, influence IGF-1R
signaling and sensitivity to IGF-1R inhibitors in ALL lymphoblasts. Selected
combination strategies aimed at inhibiting IGF-1R and related downstream
pathways represent a potential strategy for future translation into novel ALL
therapies, in particular when AMPK is one of the signaling proteins targeted in
these combinations.

Cell viability (500 μl) was determined using the Vi Cell XR system
(Beckman-Coulter), and values are expressed as a percentage relative to
those obtained in untreated controls (means ± SEM, n = 3). Synergism
was determined using the Chou's combination index (CI) based on the
following equation: CI = [(D1 combination/D1 single) +
(D2 combination/D2 single)] [63]. The numerators D1 combination and
D2 combination represent the concentration of the drug
D1 and D2, respectively, used in the combination
treatment that inhibits cell growth by x%. The denominators D1 single
and D2 single represent the concentration of drug D1 and D2
as single agent needed to achieve the same level of growth inhibition than
in the combination (x%).

GML carried out major experiments including Western blots, cell growth
proliferation and apoptosis assays, statistical data analysis, conceived the
study and participated in designing the experiments. GJL participated in data
analysis, flow-cytometry assays, and in designing the experiments. GF carried
out Western blots. JCB is the corresponding author and is responsible for
experimental design and coordination. All authors read and approved the final
manuscript.

Acknowledgements

This research was supported by grants from the Leukemia and Lymphoma Society
(grant number 6168-09), and the Micah Batchelor Award to Julio C. Barredo.